【作者】 田继远；

【导师】 陈西广； 孙修勤；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 淋巴囊肿病(Lymphocystis disease,LCD)是由虹彩病毒科(Iridoviridae)淋巴囊肿病毒(Lymphocystis disease virus, LCDV)引起的一种鱼类病毒性传染病。淋巴囊肿病在我国北方养殖牙鲆中大规模发生,给养殖者造成了重大经济损失,严重威胁我国的海水养殖业。面对淋巴囊肿病的蔓延,如何实施预防以及有效地治疗该病毒病,就成了养殖产业面临的一个迫在眉睫的课题。DNA疫苗又称基因疫苗或核酸疫苗,是将编码某种抗原蛋白的外源基因(一般是DNA)与真核表达载体质粒重组,利用某种方法直接导入动物细胞内,使带有目的基因的表达载体通过宿主细胞的表达系统合成抗原蛋白,诱导宿主产生针对该抗原蛋白的非特异性和特异性免疫应答,从而起到免疫保护作用。DNA疫苗与传统疫苗相比,制备方法简单、成本低、适合大批量生产、质粒DNA非常稳定且易于贮存和运输。更为重要的是DNA疫苗的抗原基因能在体内长期表达,不断刺激机体的免疫系统达到免疫效果。另外它可组成多价疫苗,形成对多个抗原表位的免疫保护作用。目前,水产养殖业对鱼类疾病采用的免疫途径基本上为肌肉注射方式。一般认为,注射方式产生的免疫效果强于浸浴和口服,但该方式也伴随着一些负面问题,例如较高的人力和设备成本,注射量不易控制,养殖鱼个体差异较大,偶而出现鱼类皮肤破损。对于养殖的鱼类而言,用口服疫苗免疫是一条有效的免疫途径。然而,由于各种酶的存在以及恶劣的胃肠条件,以DNA为基础的疫苗会被水解或者发生变性,这使核酸疫苗的应用受到了很大限制。为了克服这一弊端,本文根据DNA疫苗的特征,运用油包水(W/O)和水包油包水(W/O/W)乳化技术制备了三种不同的淋巴囊肿病毒微囊核酸疫苗:海藻酸盐微囊核酸疫苗;聚丙交酯乙二醇酸(PLGA)微囊核酸疫苗和壳聚糖微囊核酸疫苗。载有抗淋巴囊肿病毒核酸疫苗的海藻酸盐微囊能通过油包水乳化方式(W/O)被成功制备。这种海藻酸盐微囊的产率,载药量和包被效率分别为90.5%, 1.8%,92.7%。制备的海藻酸盐微囊直径小于10μm,而且其表面形态为圆形。傅立叶红外光谱分析显示,相对于海藻酸钠,载疫苗的海藻酸钠微囊中出现了明显的DNA单键与双键峰值。琼脂糖凝胶电泳发现,在包被过程中,有少量超螺旋质粒DNA的空间结构发生改变,被转变成开环和线性质粒。质粒DNA的累积释放试验表明:在pH = 2.0的酸性介质中,从载药微囊中释放的pDNA少于10%,而在pH = 9.0的碱性介质中有少于6.5%的释放。RT-PCR和免疫荧光试验表明,在进行口服管理之后10到90天内,pDNA表达了RNA和绿色荧光蛋白。间接法酶联免疫吸附试验表明(indirect ELISA),与裸露质粒DNA(pDNA)免疫过的鱼相对照,用载有质粒DNA(pDNA)的海藻酸盐微囊免疫牙鲆之后,从第3周到第16周的这段日期里,鱼的血清免疫响应呈阳性(O.D≥0.3)。本论文通过改良的水包油包水(W/O/W)双乳化技术,质粒DNA(pDNA)能成功地包被进聚丙交酯乙二醇酸(PLGA)胶囊里。这种胶囊的包被效率,载药百分比和产率分别是78-88%,0.5-0.7%和83.5-86.5%,它的粒径小于14μm。在模拟胃肠液及体液累积释放试验中发现,在模拟胃液中,它的累积释放能力依次为pH 2.0 > pH 9.0 > pH 7.4。裸露质粒(pDNA)与胶囊中质粒(pDNA)超螺旋比例分别为80%和89%左右,这表明在包被过程中有少量的超螺旋质粒(pDNA)发生降解。反转录技术(RT-PCR)说明,载药胶囊口服免疫后10到90天,含有主要衣壳蛋白基因(MCP gene)信息的RNA广泛存在于牙鲆各种组织内。免疫荧光照片显示:服用载药PLGA胶囊后,淋巴囊肿病毒主要衣壳蛋白在牙鲆供检组织内得到了有效表达。除此之外,间接法酶联免疫吸附试验表明,从口服后第1周到第24周血清的免疫响应呈阳性(O.D≥0.3),而口服裸露核酸疫苗后,血清的免疫响应呈阴性。基于壳聚糖在水溶液中带正电荷,核酸疫苗在水溶液带负电荷,两者能互相吸附的特征,本论文运用油包水乳化法(W/O)技术,构建了一种壳聚糖微囊核酸疫苗。这种微囊的产率,载药百分比及包被效率分别是93.6%, 0.3%和94.5%。扫描电镜照片表明,载淋巴囊肿病毒核酸疫苗壳聚糖微囊呈球形且具有光滑的外表。琼脂糖电泳分离结果显示相对于开环质粒和线性质粒疫苗(pDNA),该微囊含有高比例的超螺旋质粒,充分说明核酸疫苗在包被前后的转染效率没有大的改变。模拟胃肠环境下的体外缓释实验表明,被包被于壳聚糖微囊中的质粒疫苗(pDNA)在酸性介质中比在碱性介质中有更快的释放速度。壳聚糖微囊核酸疫苗在磷酸缓冲液(PBS,pH 7.4)中的释放曲线揭示,微囊被肠道吸收以后能持续释放的时间可以达到42天。RT-PCR和免疫荧光结果表明,用淋巴囊肿病毒壳聚糖微囊核酸疫苗对牙鲆进行口服免疫之后,在10-90天里,均能检测到包含淋巴囊肿病毒主要衣壳蛋白(MCP)遗传基因信息的RNA存在于鱼的各供检组织内,在组织切片中同时也发现含有荧光表达蛋白的抗原蛋白被核酸疫苗表达。除此之外,用口服淋巴囊肿病毒壳聚糖微囊核酸疫苗免疫牙鲆之后,显示了抗体浓度明显增加,其免疫响应呈阳性(O.D > 0.3)。从免疫效果来分析, PLGA微球疫苗效果最好,但PLGA价格相对另外两种材料稍贵,加工工艺相对复杂,口服管理前要保证有机溶剂挥发完全。壳聚糖微球疫苗与海藻酸盐微球疫苗均采用油包水(W/O)技术制备,前者免疫效果要优于后者,主要是因为壳聚糖本身带正电荷,核酸疫苗带负电荷,它们两者本身发生吸附,所以壳聚糖会提供更好的缓释和保护作用。本实验结果表明,海藻酸盐微囊,PLGA微胶囊及壳聚糖微囊均是理想而富有前景的药物载体。这些包被技术因为易于操作,原料价格低廉以及显著的免疫功效而具有广阔的前景,它们有可能在药物传输方面得到广泛的应用。更多还原

【Abstract】 Lymphocystis disease, caused by lymphocystis disease virus (LCDV), is a kind of infectious disease of many fish. The pathogen was suggested as an iridovirus based on histopathological and morphological evidences. Outbreaks of lymphocystis disease with high mortalities have been reported in cultured Japanese flounder in Northern culture of China and caused significant economic losses in some marine net-cage farms. Therefore, in order to control LCDV, it is essential to find a vaccine and vaccinate fish by optimal approach.DNA vaccines are plasmid DNAs encoding specific proteins that can be expressed in cells of an inoculated host. The plasmids used are eukaryotic expression vectors, which contain the necessary elements for expression in eukaryotic cells. DNA vaccines are considered to be a promising form of vaccine for the control of infectious diseases and cancers since they offer many advantages over other conventional vaccines such as peptide or attenuated live pathogens. For instance, DNA vaccines are relatively safe and can be administered repeatedly without adverse effects. In addition, DNA vaccines are comparatively easy to produce on a large scale and are able to yield products with high purity and stability. Most importantly, effective DNA vaccine delivery systems, such as direct intradermal administration of DNA vaccines via gene gun to professional antigen presenting cells (APCs), have been well established. Experts in the research field now believe that the first step in the success of nucleic acids as a biopharmaceutical drug could come in terms of genetic immunization (DNA vaccines) which only needs transient expression of the encoding protein unlike long term expression in gene therapy. Plasmid DNA encoding for a variety of antigens (proteins) have been demonstrated in animal models of infectious diseases, cancers and allergies.Nucleic acid-based immunotherapy is a new treatment option for many complex disorders including a variety of infectious diseases and cancers. More importantly, DNA-based vaccines could have increased advantages over recombinant proteins and peptides in treatments of viral and parasitic infectious diseases as well as cancers. The reproducible and robust clinical efficacy of naked pDNA in fish trials has been demonstrated by intramuscular, intravenous, or intradermal injection. However, for fry in the aquaculture industry, injection approach is inappropriate because of the difficulty in operation. In addition, injection may harm the skin of fish, and bacterium and/or parasite would infect fish and cause higher mortality rate. The lower efficacy of orally administered pDNAs may be attributable to the inactivation of pDNA caused by the nucleases, lipases and peptidases present in the gastrointestinal tract. The pDNA encapsulated in microparticles can be protected from degradation in the gut, and a controlled release of pDNA could be achieved. In order to overcome the problems described above, three kinds of microparticles, including alginate microspheres loaded with DNA vaccine, PLGA microcapsules loaded with DNA vaccine and chitosan microspheres loaded with DNA, were prepared to vaccinate Japanese flounder.Oral alginate microcapsules loaded with pDNA against fish lymphocystis disease viruses (LCDV) were prepared with a modified oil containing water (W/O) emulsification method. Yield, loading percent and encapsulation efficiency of alginate microcapsules were 90.5%, 1.8% and 92.7%, respectively. Alginate microspheres prepared had size diameters of less than 10μm, and their shape was spherical. As compared to sodium alginate, a remarkable increase of DNA-phosphodiester and DNA-phosphomonoester bonds was observed for alginate microcapsules loaded with pDNA by Fourier transform infrared (FTIR) spectroscopic analysis. Agarose gel electrophoresis showed a little supercoiled pDNA was transformed to open circular and linear pDNA during encapsulation. The cumulative release of pDNA in alginate microcapsules was≤10% in pH 2.0 acidic media, and it was less than 6.5% in pH 9.0 alkaline media in 12 h. RT-PCR and immunofluorescence image indicated that pDNA expressed RNA and fluorescence protein in tissues of fish 10–90 days after oral administration. An indirect enzyme-linked immunosorbent assay (ELISA) showed that immune response of sera was positive (O.D≥0.3) from week 3 to week 16 for fish vaccinated with alginate microcapsules loaded with pDNA, in comparison with fish vaccinated with naked pDNA.Poly(DL-lactide-co-glycolide) (PLGA) microcapsules, loaded with plasmid DNA (pDNA) against lymphocystis disease virus (LCDV), were prepared by modified water in oil in water (W/O/W) double emulsion method in our laboratory. Encapsulation efficiency, loading percent and diameter of microcapsules were 78-88%, 0.5-0.7% and less than 10μm, respectively. In simulated gastric fluid (SGF), less than 10% of pDNA was released from microcapsules in 12 h, and about 6.5% of pDNA was released in 12 h in simulated intestinal fluid (SIF). The content of the supercoiled of pDNA in microcapsules and control was 80% and 89% respectively, which indicated that a little supercoiled pDNA degradation occurred during encapsulation. RT-PCR showed that lots of RNA containing information of MCP gene existed in all tissues of fish vaccinated with microcapsules 10-90 days after oral administration. Immunofluorescence images displayed that major capsid protein (MCP) of LCDV was expressed in tissues of fish vaccinated with pDNA-loaded microcapsules. In addition, indirect enzyme-linked immunosorbent assay (ELISA) showed that the immune responses of sera were positive (O.D≥0.3) from week 1 to week 24 for fish vaccinated with microcapsules, in comparison with fish vaccinated with naked pDNA.Chitosan microcapsules containing a plasmid vaccine (pDNA) against lymphocystis disease virus were prepared through an emulsion-based methodology. The yield, drug loading and encapsulation efficiency of microcapsules were 93.6%, 0.3% and 94.5%, respectively. Scanning electron microscopy (SEM) showed that pDNA-loaded microcapsules yielded a spherical shape with smooth surfaces. The disproportion of supercoiled to open circle and linear pDNA suggested that high transfection efficiencies of pDNA in microcapsules were retained. In simulated gastrointestinal tract environment, the release rate of pDNA encapsulated in acidic media was faster than that in alkaline media. The release profile at PBS buffer (PH 7.4) displayed that pDNA-loaded chitosan microcapsules had a sustained release up to 42 days after intestinal imbibition. In addition, the results of RT-PCR and immunofluorescence images indicated that the pDNA expressed antigen protein in tissues of fish 10-90 days after oral administration.Our results suggested that pDNA-loaded alginate microcapsules and PLGA microcapsules as well as chitosan microcapsules were promising carriers for pDNA vaccine. The three pDNA-loaded microcapsules have potential for drug delivery applications due to their ease of operation, low cost and notable immunisation efficacy.更多还原